Lipoproteins and Inflammation in the Development of Diabetic Complications

在过去的几年里，我们在两个大的1型和2型糖尿病队列中清楚地证明了这一点 (DCCT/EDIC和VADT队列)高水平的含有氧化型低密度脂蛋白(OxLDL)的免疫复合体(IC) 晚期糖化终产物修饰的低密度脂蛋白(AGE-LDL)与冠状动脉疾病有关 (CAD)1-4，并能很好地预测1型早期CAD的发展和进展 糖尿病3.我们还在VADT队列中显示，其中包括许多晚期冠心病患者， IC中高水平的丙二醛修饰的低密度脂蛋白能够预测急性心血管疾病 (CVD)事件4.然而，当循环IC同时含有高水平的丙二醛-低密度脂蛋白和高水平的氧化低密度脂蛋白或 AGE-低密度脂蛋白患急性事件的风险降低，这使我们得出结论，修饰的脂蛋白 IC携带在斑块进展/稳定和斑块失稳过程中起着差异性调节作用。 我们的机制研究支持了上述假设。氧化低密度脂蛋白IC诱导巨噬细胞转化 进入泡沫细胞5-7，导致促生存基因8上调，并诱导促炎因子的释放 细胞因子10。氧化低密度脂蛋白IC还能诱导系膜细胞产生更多的胶原。相比之下，丙二醛-低密度脂蛋白 IC诱导巨噬细胞凋亡，刺激基质金属蛋白酶表达 巨噬细胞和主动脉内皮细胞，并减少系膜细胞胶原基因的表达。 假设和具体目的：根据我们的临床和体外初步数据，我们假设 循环IC中低密度脂蛋白的主要修饰类型(丙二醛-低密度脂蛋白、ox低密度脂蛋白或年龄-低密度脂蛋白)具有显著的 斑块进展/不稳定的影响。我们假设，当丙二醛是主要的 IC携带的低密度脂蛋白，巨噬细胞摄取这些IC诱导细胞凋亡，增加释放 MMPs和胶原酶活性增加，从而导致斑块不稳定。相比之下，当 氧化低密度脂蛋白是低密度脂蛋白在IC中的主要修饰，这些IC的摄取诱导巨噬细胞存活和 刺激促炎介质和生长因子的释放，从而促进斑块的形成 扩展/稳定。此外，我们假设包括丙二醛-低密度脂蛋白IC和MMPs的生物标志物小组将是 比一组传统的心血管疾病更能有力地预测高危患者发生急性心血管事件 生物标志物。 为了验证上述假设，我们提出了三个目标。第一个目标是比较两个基因的激活模式 巨噬细胞暴露于IC(丙二醛-LD-IC和oxLDL-IC)，这些IC对巨噬细胞存活和凋亡的影响 巨噬细胞，以及涉及的途径。在第二个目标中，我们将测量已知的新生物标记物 与斑块不稳定性相关的患者样本来自VADT队列，在该队列中，MDA-LDL IC和 常规的CVD生物标志物以前已经被测量过了。我们将确定一个小组是否包括 丙二醛-低密度脂蛋白IC、基质金属蛋白酶-1和基质金属蛋白酶-9对高血压病患者的预测能力显著提高。 急性心肌梗塞的风险。最后，在第三个目标中，我们将在动脉粥样硬化的动物模型上确定 治疗是否阻断/减少oxLDLIC和MDA-LDLIC对Fc？Rs的结合 与小鼠IgM、抗oxLDL和IgM、抗MDA-LDL一起，可防止斑块的进展和不稳定性。 方法：细胞分离培养、脂蛋白分离修饰、抗体、蛋白质分离 阵列，细胞存活率/凋亡检测，RT-PCR，免疫印迹，酶活性检测，免疫组织化学 动脉粥样硬化病变的基因表达分析和组织学分析。 与退伍军人管理局健康和意义的相关性：对造成以下差异的机制的定义 不同修饰的低密度脂蛋白诱导的细胞活化、存活和凋亡可能允许定义新的 遏制心血管疾病进展的治疗目标和新的预防策略 2型糖尿病患者的并发症和急性血管事件的发生。

In the past few years we have clearly demonstrated in two large diabetic cohorts of type 1 and type 2 diabetes (DCCT/EDIC and VADT cohorts) that high levels of immune complexes (IC) containing oxidized LDL (oxLDL) and advanced glycated end-products-modified LDL (AGE-LDL) are associated with coronary artery disease (CAD)1-4 and can strongly predict the development and progression of CAD in the early stages of type 1 diabetes3. We have also shown in the VADT cohort, which includes many patients with advanced CAD, that high levels of malondialdehyde-modified (MDA)-LDL in IC are able to predict acute cardiovascular disease (CVD) events4. However, when circulating IC contain both high levels of MDA-LDL and high levels of oxLDL or AGE-LDL the risk to suffer an acute event is reduced4 leading us to conclude that the modified lipoproteins carried by IC play a differential modulating role in plaque progression/stability and plaque destabilization. Our mechanistic studies support the above hypothesis. OxLDL IC induce the transformation of macrophages into foam cells5-7, lead to upregulation of pro-survival genes8 and induce the release of pro-inflammatory cytokines10. OxLDL IC also induces increased collagen production by mesangial cells12.. In contrast, MDA-LDL IC induce macrophage apoptosis, stimulate the expression of matrix metalloproteinases (MMPs) by macrophages and by aortic endothelial cells, and reduce the expression of collagen genes in mesangial cells. Hypothesis and Specific Aims: Based on our clinical and in vitro preliminary data, we hypothesize that the type of predominant modifications of LDL in circulating IC (MDA-LDL, oxLDL or AGE-LDL) has a significant impact in plaque progression/destabilization. We postulate that, when MDA is the predominant modification of the LDL carried by IC, the uptake of those IC by macrophages induces cell apoptosis, increased release of MMPs, and increased collagenase activity, thus contributing to plaque destabilization. In contrast, when oxLDL is the predominant modification of LDL in IC, the uptake of these IC induce macrophage survival and stimulate the release of pro-inflammatory mediators and growth factors, thus contributing to plaque expansion/stability. Furthermore we postulate that a biomarker panel including MDA-LDL IC and MMPs will be more able to strongly predict patients at high risk to suffer acute CVD events than a panel of conventional CVD biomarkers. To test the above hypotheses we propose three aims. The first aim will compare the activation patterns of macrophages exposed to IC (MDA-LD IC and oxLDL IC), the effect of these IC on the survival and apoptosis of macrophages, and the pathways involved. In the second aim we will measure novel biomarkers known to be associated with plaque instability in patient samples from the VADT cohort in which MDA-LDL IC and conventional CVD biomarkers have been previously measured. We will determine whether a panel including MDA-LDL IC, MMP-1 and MMP-9 will have a significantly higher predictive power to identify patients at high risk for acute MI. Finally in the third aim we will determine, on an animal mouse model of atherosclerosis and type 2 diabetes whether blocking/reducing engagement of Fc¿Rs by oxLDL IC and MDA-LDL IC by treatment with mouse IgM anti-oxLDL and IgM anti-MDA-LDL will prevent plaque progression and instability. Methods: Cell isolation and culture, separation and modification of lipoproteins, isolation of antibodies, protein arrays, cell viability/apoptosis assays, rtPCR, western blots, enzyme-activity assays, immunohistochemical analysis of gene expression and histologic analysis of atherosclerotic lesions. Relevance to VA Health and Significance: The definition of mechanisms responsible for the differences in cell activation, survival and apoptosis induced by different modified LDL-IC may allow to define new therapeutic targets and new prevention strategies that will curtail the progression of cardiovascular complications and the occurrence of acute vascular events in patients with type 2 diabetes.